Cancer incidence and mortality have not declined at the same rate as other major causes of death. This suggests that primary prevention, including the use of chemopreventative agents, is a valuable approach to decreasing mortality, as it avoids all the potential shortfalls and difficulties associated with cancer therapy.
Selenium is an essential nonmetallic nutrient in mammals. Its deficiency is associated with increased incidence of leukemia and cancers of bladder, breast, gastrointestinal tract (e.g., colon and rectum), ovary, stomach, lung and prostrate. There is convincing evidence that a dietary supplement of selenium substantially reduces the incidence of a wide variety of cancers in laboratory animals. Higher intake of selenium provides protection by correcting nutritionally deficient status in animals and also increases antitumorigenic activity. El-Bayoumy, Cancer Res., 45:3631-3635 (1985); Reedy et al., Cancer Res. 47:5901-5904 (1987); Nayini et al., Carcinogenesis 10:509-512 (1989); El-Bayoumy et al., Cell. Biochem., 22:92-100 (1995); El-Bayoumy, xe2x80x9cThe role of selemium in cancer prevention,xe2x80x9d in: Cancer Principles and Practice of Oncology, DeVita et al., eds., J.B. Lippincot Co., Phildelphia (1991).
Epidemiological studies in humans also suggest a protective role for selenium in human cancers. Human cancer mortality is lower in areas providing an adequate dietary intake of selenium as estimated from the selenium content in grains and forage crops in various regions of the United States, or the dietary selenium intake as calculated from food consumption data in various countries. Schrauzer et al. Bioinorg. Chem. 7:23-31 (1977).
An excess of selenium is associated with toxicity and a large number of health disorders, such as body weight loss, liver damage, splenomegaly, pancreatic enlargement, anemia, hair loss and abnormal nails. The margin between its protective role and adverse effect is very low and depends on the form of selenium being used. For its protective role, the rate of release of selenium from its compound to the selenium pool should be in the range of its required rate of absorption by the biological system.
The search for the best form of selenium for cancer chemoprevention has led to the investigation of a wide variety of selenium forms. Examples of compounds that have been investigated include inorganic salts like sodium selenite, as well as organoselenium compounds such as selenocysteine (a naturally occurring form of selenium) and a number of structurally designed aliphatic and aromatic selenium-containing compounds such as heptylselenocyanate and pentylselenocyanate. Ip et al., Carcinogenesis 16:35-38 (1995).
Although inorganic selenium compounds inhibit carcinogenesis, they are toxic. Organic sources of selenium that have been investigated to date, such as the naturally occurring selenium-containing amino acids selenomethionine and selenocysteine which are ingested via cereals, vegetables, and grains, are somewhat more effective than inorganic selenium in cancer prevention but have comparable toxicity. El-Bayoumy, xe2x80x9cThe role of selenium in cancer prevention,xe2x80x9d in: Cancer Principles and Practice of Oncology, DeVita et al., eds., J. B. Lippincot Co., Philadelphia (1991).
Among the organoselenium compounds investigated to date, the benzene-based compound 1,4-phenylenebis(methylene)selenocyanate (also known as p-xyleneselenocyanate, or p-XSC) is reported to exert the most effective chemopreventative effect on chemically induced carcinogenesis in the mammary glands, colon, and lung of laboratory animals. Reddy et al., Cancer Res. 52:5635-5640 (1992); Reddy et al., J. Natl. Cancer Inst. 89:506-512 (1997); El-Bayoumy et al., Cancer Res. 52:2402-2407 (1992); El-Bayoumy et al., Carcinogenesis 14:1111-1113 (1993). Organic compounds having a 5-membered heterocyclic ring substituted with an alkylene isothiocyanate, wherein the heteroatom is sulfur, selenium, or oxygen, have also been described as having cancer chemopreventative activity (U.S. Pat. No. 6,166,003, Lam).
The invention provides novel organoselenium compounds for use in cancer chemoprevention. In one embodiment, the compound of the invention is a 5-membered heterocyclic ring substituted with an alkylene selenocyanate at least at the 2-position on the ring, as shown in formula I: 
wherein:
R1 is H, (alkylene)-SeCN, or a blocking group;
R2 is H, (alkylene)-SeCN, or a blocking group;
R3 is H, (alkylene)-SeCN, or a blocking group; and
R4 is (alkylene)-SeCN; and
Z is S, Se or O.
Also provided by the invention are cysteine-containing conjugates of the organoselenium compound shown in formula I, wherein the cyanate group (xe2x80x94CN) at one or more of R1, R2, R3 and R4 is replaced with a cysteine or a cysteine-containing peptide such as the tripeptide glutathione (xcex3-glutamylcysteinylglycine). Preferably, the sulfur atom of the cysteine residue is covalently linked to the selenium atom of the at least one (alkylene)-SeCN moiety. A preferred conjugate is the compound of formula I wherein the cyanate group (xe2x80x94CN) of at least one (alkylene)-SeCN moiety is replaced with a cysteine, an N-acetyl cysteine, a cysteinylglycine, or the tripeptide glutathione.
Preferably, in compounds wherein R1=R4=methylene-SeCN, at least one of R2 and R3 is not H. However, in pharmaceutical and food additive compositions and in methods of use of the cancer chemopreventive compounds of the invention, the compound can include formula I wherein R1=R4=methylene-SeCN and R2=R3=H.
In another embodiment, the compound of the invention is a 5-membered heterocyclic ring substituted with an alkylene isoselenocyanate at least at position 2 on the ring, as shown in formula II: 
wherein:
R1 is H, (alkylene)-NCSe, or a blocking group;
R2 is H, (alkylene)-NCSe, or a blocking group;
R3 is H, (alkylene)-NCSe, or a blocking group;
R4 is (alkylene)-NCSe; and
Z is S, Se or O.
Also provided by the invention are cysteine-containing conjugates of the organoselenium compound shown in formula II, wherein the isoselenocyanate group (xe2x80x94NCSe) at one or more of R1, R2, R3 and R4 is replaced with a cysteine-containing moiety having the general formula xe2x80x94N(H)C(Se)R5 wherein R5 is cysteine, a derivative thereof, or a peptide containing cysteine. For example, one or more of R1, R2, R3 and R4 is replaced with xe2x80x94N(H)C(Se)cysteine or xe2x80x94N(H)C(Se)glutathione where R5 is cysteine or glutathione, respectively. Preferably, the sulfur atom of the cysteine residue is covalently linked to the carbon atom of the at least one (alkylene)-NCSe moiety. A preferred conjugate is the compound of formula II wherein the isoselenocyanate group (xe2x80x94NCSe) of at least one (alkylene)-NCSe moiety is replaced with xe2x80x94N(H)C(Se)cysteine, xe2x80x94N(H)C(Se)xe2x80x94N-acetylcysteine, xe2x80x94N(H)C(Se)cysteinylglycine, or xe2x80x94N(H)C(Se)glutathione.
In yet another embodiment, the compound of the invention is a xyleneisoselenocyanate compound as shown in formula III: 
wherein:
R1 is H, (alkylene)-NCSe, or a blocking group; and
R2 is (alkylene)-NCSe.
The disubstituted form of the compound of formula III, 1,4-phenylenebis(alkylene)selenocyanate, (also known as p-xyleneisoselenocyanate, or p-XISC) is especially preferred.
Also provided by the invention are cysteine-containing conjugates of the organoselenium compound shown in formula III, wherein the isoselenocyanate group (xe2x80x94NCSe) at one or both of R1 and R2 is replaced with a cysteine-containing moiety having the general formula xe2x80x94N(H)C(Se)R3 wherein R3 is cysteine, a derivative thereof, or a peptide containing cysteine. For example, one or both of R1 and R2 is replaced with xe2x80x94N(H)C(Se)cysteine or xe2x80x94N(H)C(Se)glutathione where R3 is cysteine or glutathione, respectively. Preferably, the sulfur atom of the cysteine residue is covalently linked to the carbon atom of the at least one (alkylene)-NCSe moiety. A preferred conjugate is the compound of formula III wherein the isoselenocyanate group (xe2x80x94NCSe) of at least one (alkylene)-NCSe moiety is replaced with xe2x80x94N(H)C(Se)cysteine, xe2x80x94N(H)C(Se)xe2x80x94N-acetylcysteine, xe2x80x94N(H)C(Se)cysteinylglycine or xe2x80x94N(H)C(Se)glutathione.
The term xe2x80x9calkylenexe2x80x9d as used herein means a divalent saturated hydrocarbon chain containing one or more carbon atoms. An alkylene can be linear or branched. Examples of linear alkylenes include methylene (xe2x80x94CH2xe2x80x94), ethylene (xe2x80x94CH2xe2x80x94CH2xe2x80x94), propylene (xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94), butylene (xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94) and so on. Examples of branched alkylenes include compounds such as xe2x80x94CH2xe2x80x94CH(CH3)-CH2xe2x80x94, xe2x80x94CH2xe2x80x94CH(CH2CH3)-CH2xe2x80x94, CH2xe2x80x94CH(CH3)-CH2xe2x80x94CH(CH3)-CH2xe2x80x94, xe2x80x94CH2xe2x80x94C(CH3)2xe2x80x94CH2xe2x80x94and the like. The compound of the invention is not intended to be limited by the length of the alkylene chain that connects a selenocyanate (xe2x80x94SeCN) or an isoselenocyanate (xe2x80x94NCSe) group (or conjugate thereof) to the heterocyclic moiety.
Moreover, where the compound contains an alkylene selenocyanate or alkylene isoselenocyanate substituent (or conjugate thereof) at more than one position on the heterocyclic ring, the alkylene functionalities connecting the selenocyanate or isoselenocyanate groups (or conjugates thereof) at the respective ring positions can have different numbers of carbon atoms.
Linear alkylene chain lengths of about 1 to about 20 carbons (C 1-C 20) are preferred for ease of synthesis.
Preferably, the alkylene chain length connecting the selenocyanate or isoselenocyanate group (or conjugate thereof) to the heterocyclic moiety is about 1 to about 12 carbons (C 1-C 12), more preferably about 1 to about 6 carbons (C 1-C 6), most preferably about 1 to about 4 carbons (C 1-C 4). For example, a representative compound of the invention is 2-thienyl methyl isoselenocyanate, which has a C, alkylene functionality (i.e., a 1 carbon chain) connecting isoselenocyanate (-NCSe) to the 2-position of the thienyl ring (II, wherein R1=R2 =R3 =H and R4 is (methylene)-NCSe; and Z =S).
As used herein, the term xe2x80x9cheterocyclic ringxe2x80x9d refers to a ring structure that contains as a member of the ring at least one noncarbon atom. In the heterocyclic ring of the compound of formula I or formula II, or conjugates thereof, the non-carbon member Z is preferably S, but can alternatively be Se or O.
Where one or more of R1, R2 and R3 is a blocking group, it is not intended to be limited to any particular blocking group, and it should be understood that different blocking groups can be used at different ring positions. A blocking group can be aromatic or aliphatic, and can be linear, branched, or cyclic. A blocking group is preferably an alkyl, alkoxy, aryl, aryloxy, alkylmercapto, alkylene aryl, CX3, or X; wherein X is F, Cl, or Br. Aromatic or aliphatic constituents of a blocking group can be substituted or unsubstituted. More preferably the blocking group is CH3, OCH3, SCH3, CX3 or X; most preferably it is OCH3, SCH3, CF3 or F.
Substituting the 5-position of the ring is desirable in order to enhance the chemopreventive effect of the compound of the invention, for example by slowing metabolism of the ring moiety in a treated subject or by way of a steric effect. Therefore, the compound of formula I, II or III, wherein R1 is (alkylene)-SeCN (in the case of formula I) or (alkylene)-NCSe (in the case of formula II and formula III), cysteine-containing conjugates of either (alkylene)-SeCN or (alkylene)-NCSe, or a blocking group is especially preferred.
Also included in the present invention are methods for making a compound having formula I, formula II, or formula III,-as well as cysteine-containing conjugates thereof.
The present invention further includes a pharmaceutical composition comprising an active ingredient which is a compound of the present invention. Preferred embodiments of the pharmaceutical composition are those that contain preferred embodiments of the compound having formula I, II, or III, or cysteine-containing conjugates thereof, as set forth above. Included in the pharmaceutical composition is, preferably, a pharmaceutically acceptable carrier, which can comprise a pharmaceutically acceptable salt. The pharmaceutical composition is suitable for treatment of an existing condition or for prophylactic use.
Additionally, the compound of the present invention can be incorporated into food or drink as an additive or supplement, or formulated for cosmetic use, as in a body lotion, creme, sunscreen or the like.
The present invention further includes a method for preventing the occurrence or progression of a cancer or a precancerous condition, including cellular changes characterized by neoplasia. The method comprises administering to a mammal a chemopreventive composition comprising the compound the present invention in an amount effective to prevent the occurrence of cancer (carcinogenesis) or a precancerous condition, or to slow or halt the progression of cancer or precancerous conditions. The chemopreventive composition can be administered as a therapeutic to treat an existing condition or as a prophylactic in advance of exposure to a carcinogenic compound or event.